Lipid molecules act not only as energy resources or physical barriers, they are also actively involved in regulating cellular signaling, membrane trafficking, and the transcriptional network. Lipid metabolic dysfunction contributes to the development of various human diseases. Fatty acids are building blocks and precursors of all lipid molecules and have crucial impact on cell physiology and pathology. Excess accumulation of saturated fatty acids in non-adipose tissues leads to cytotoxicity and the pathogenesis of nonalcoholic fatty liver diseases, cardiomyopathy and type II diabetes mellitus; whereas unsaturated fatty acids are less toxic and even exert protective effects. Recent studies suggest that differences of fatty acids in lipotoxicity may be associated with their distinct efficiency duing the incorporation into lipid droplets (LDs). However the underlying molecular mechanisms governing this LD incorporation heterogeneity between different fatty acid molecules remain poorly understood. In our recent studies, we have developed a new chemical imaging strategy for tracking the spatiotemporal dynamics of specific lipid molecules at the subcellular resolution in living cells and organisms, and directly visualized the difference between saturated and unsaturated fatty acids during their incorporation into LDs. This proposal aims to dissect the molecular mechanisms that regulate LD incorporation heterogeneity between different fatty acid molecules, and will combine innovative technology development and detailed mechanistic characterization. Aim 1 will develop a high-throughput imaging/sorting microfluidic platform based the new chemical imaging microscopy. Aim 2 will elucidate new genes and pathways in regulating fatty acid metabolic dynamics through high-throughput forward genetic screens. We have established unique collaborations with scientists for this project. Successful accomplishment of the proposed studies will provide a new technology platform for whole-organism microscopic screening, and provide new insights into fatty acid metabolism and their physiological and pathological functions.